仔猪血清大豆抗原蛋白抗体间接ELISA检测方法的建立  

Establishment of an indirect enzyme-linked immune sorbent assay for detection of serum antibody of soybean antigen protein in piglets

作　　者：寇亚楠[1] 马良友[1] 罗莹[1] 王希春[1] 吴金节[1] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《西北农林科技大学学报（自然科学版）》2014年第3期17-22,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：科技部科技富民强县专项行动计划项目(国科发农【2012】745号)

摘　　要：【目的】研究并建立大豆抗原蛋白血清抗体间接ELISA检测方法。【方法】首先用大豆抗原蛋白对仔猪进行致敏,然后采用棋盘滴定法确定最佳抗原包被质量浓度及血清稀释倍数,并对其他条件进行优化,最终建立检测血清大豆抗原蛋白11S、7S抗体的间接ELISA方法,确立待测血清判定标准,并对建立的ELISA方法进行重复性、准确性和敏感性检测。【结果】建立的ELISA方法的最佳反应条件为:11S抗原蛋白最适包被质量浓度为2.0μg/mL,包被作用时间37℃下2h;将10g/L BSA作为封闭液,37℃封闭1h;待测血清37℃作用1h;酶标二抗最适稀释度为1∶1 500,37℃作用1h;显色剂最佳显色时间15min。7S抗原蛋白的最适包被质量浓度为2.5μg/mL,包被作用时间37℃下2h;将10g/L BSA作为封闭液,37℃封闭1h;待测血清37℃作用1h;酶标二抗最适稀释度为1∶1 500,37℃作用1h;显色剂最佳显色时间25min。11S和7S抗原蛋白间接ELISA检测方法的批内、批间变异系数均小于10%,重复性较好,灵敏度较高。【结论】建立了仔猪血清大豆抗原蛋白抗体间接ELISA检测方法,该法具有很强的特异性和敏感性,可用于大豆抗原蛋白11S、7S过敏反应的临床诊断。[Objective] The experiment was conducted to establish an indirect enzyme-linked immune sorbent assay （ELISA） to detect the serum antibody levels in piglets sensitized by soybean antigen protein. [Method] After the piglets were sensitized with 11S or 7S,the serum samples were collected and the reac- tive conditions were optimized. The optimal coating mass concentrations of antigens and dilution times of sera were determined using board titration method. An indirect enzyme-linked immune sorbent assay to de- tect the serum contents of 11S and 7S in piglets was then established and its repeatability, accuracy and sensitivity were detected. [Result] The optimal conditions of ELISA 11S were：the coating mass concentra- tion was 2.0 /~g/mL,the action time of the coating at 37 ℃ was 2 h;using 10 g/L BSA as sealing fluid for 1 h at 37 ℃ ;the test serum worked for 1 h at 37 ℃ ,the enzyme labeled antibody was diluted into 1-1 500 and the reactive time was 1 h at 37 ℃,and the reaction time of substrate was 15 min. The optimal condi- tions of ELISA 7S were.the coating mass concentration was 2.5 μg/mL and the action time of the coatingat 37 ℃was 2 h,using 10 g/L BSA as sealing fluid for 1 h at 37 ℃ ~the test serum worked for 1 h at 37 ℃, the enzyme labeled antibody was diluted into 1 ： 1 500 and the reaction time was 1 h at 37 ℃,and the reac- tion time of substrate was 25 min. Both intra-assay and inter-assay variation coefficients were less than 10%. [Conclusion] The established indirect ELISA for detection of serum antibody levels in piglets sensi- tized by soybean antigen protein had good repeatability and high sensitivity, and could be used in clinical di- agnosis of allergy induced by soybean protein antigen.

关 键 词：大豆抗原蛋白 仔猪 血清抗体 间接ELISA 

分 类 号：S852.43[农业科学—基础兽医学]